Xanthones from the Roots of Polygala caudata and their Antioxidation and Vasodilatation Activities in vitro

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Three new xanthones, 2-hydroxy-1,6,7-trimethoxyxanthone (1), 1,4-dimethoxy-2,3-methylenedioxyxanthone (2), and 7-hydroxy-1,2-dimethoxyxanthone (3), together with five known compounds, 2,7-dihydroxy-1-methoxyxanthone (4), 1-methoxy-2,3-methylenedioxyxanthone (5), 7-hydroxy-1-methoxyxanthone (6), euxanthone (1,7-dihydroxyxanthone) (7), and gentitein (1,3,7-trihydroxyxanthone) (8), were isolated from the roots of Polygala caudata. Their structures were established on the basis of spectral evidence. In the antioxidation activity screening in vitro with luminol chemiluminescence methods, compounds 1 - 5 and 7 and 8 showed H₂O₂ scavenger activity, with a scavenging effect of 58.4 - 94.5 % at 10 μg/mL, and 26.0 - 84.7 % at 2 μg/mL. Compounds 4 and 8 also exhibited scavenging effects on the reactive oxygen free radicals produced by macrophage respiratory bursts, with a scavenging effect of 71.7 % and 63.4 % at 10 μg/mL, 41.2 % and 47.8 % at 2 μg/mL, respectively. In the vasodilatation assay, compounds 4 - 7 exhibited relaxing activity on the contractions evoked by KCl in Wistar rat thoracic aorta rings in a dose-dependent manner.

Polygala caudata Rehd. et Wils. (Polygalaceae) is a well-known Chinese medicinal plant, mainly distributed in southwestern China, and used as an expectorant and sedative agent in traditional Chinese medicine. In previous studies, 17 compounds, including 13 xanthones, had been isolated from this plant [1], [2], [3], [4], [5], but only little work on the biological activity has been reported, except for induction of the differentiation of neuroblastoma cells [6]. In our studies on the bioactive constituents, the EtOAc extract of this plant was investigated in terms of chemistry and pharmacological activities. Three new xanthones, 2-hydroxy-1,6,7-trimethoxyxanthone (1), 1,4-dimethoxy-2,3-methylenedioxyxanthone (2), and 7-hydroxy-1,2-dimethoxyxanthone (3), together with five known ones, were isolated. Here, we describe the isolation and structure elucidation of new compounds, as well as the antioxidation and vasodilatation activities in vitro of all isolated compounds.[*]

Compound 1 was obtained as white needles, and the HR-EI-MS indicated the molecular formula C₁₆H₁₄O₆ (m/z = 302.0787). Its UV spectrum showed the characteristic absorptions of xanthone at 244, 322 and 370 nm, and there was no bathochromic shift with the addition of NaOAc, which indicated the absence of 3- or 6-hydroxy moieties in 1 [7]. The IR absorption at 1650 cm^-1 revealed the presence of a conjugated carbonyl group. The ¹H-NMR spectrum showed a hydroxy (δ = 6.01, 1H, s) and three methoxy groups (δ = 4.05, 4.01 and 3.99, each 3H, s), along with four aromatic proton signals. In the ¹³C-NMR spectrum, the signal at δ = 62.7 was indicative of di-ortho-substituted methoxy groups [8]. In the NOESY spectrum (in CDCl₃), the signal at δ = 6.01 (OH-2) showed an NOE correlation with δ = 4.06 (OMe-1), while the signal at δ = 6.86 (H-5) correlated with δ = 4.01 (OMe-6), and the signal at δ = 7.65 (H-8) with δ = 3.99 (OMe-7). These indicated that the hydroxy group was linked to C-2, and the three methoxy moieties to C-1, C-6 and C-7, respectively. From the above data, compound 1 was identified as 2-hydroxy-1,6,7-trimethoxyxanthone.

Compound 2 was obtained as white needles, and its molecular formula was determined to be C₁₆H₁₂O₆ by means of HR-EI-MS (m/z = 300.0656). The UV and IR spectra of 2 were very similar to those of 1. In comparison with the ¹H-NMR spectrum of 1, that of 2 showed one methylenedioxy proton signal (δ = 6.09, 2H, s), two methoxy proton signals, and four ortho-coupled proton signals of an aromatic ring. The ¹³C-NMR spectrum of 2 showed two signals of di-ortho-substituted methoxy groups at δ = 61.4 and 61.3. The methylenedioxy moiety and the two methoxy moieties were assigned based on the HMBC spectrum (Fig. [1]). Thus, the structure of 2 was identified as 1, 4-dimethoxy-2,3-methylenedioxyxanthone.

Compound 3 was obtained as yellow needles, and its molecular formula was elucidated to be C₁₅H₁₂O₅ from HR-EI-MS (m/z = 272.0688). The UV, IR and ¹H, ¹³C-NMR spectra were similar to those of 1. The NOESY spectrum indicated that hydroxy group was at the C-7 position, and the two methoxy groups were linked to C-1 and C-2. Thus, the structure of compound 3 was determined to be 7-hydroxy-1,2-dimethoxyxanthone.

In the course of the antioxidation activity investigation in vitro, compounds 1 and 4 exhibited obvious H₂O₂ scavenger activity, their scavenging effects were > 90 % at the concentration of 10 μg/mL, while others showed moderate effects (see Table [1]). Compounds 4 and 8 could also eliminate the reactive oxygen free radicals produced by macrophage respiratory burst, and their activities were much stronger than that of ascorbic acid (see Table [2]). Besides the antioxidation activity mentioned above, compounds 4 - 7 also showed vasodilatation activity in a dose-dependent manner in the primary assay, while their activities were weak in comparison with the positive control - Verapamil, a vasodilatation drug (see Fig. [2]). These activities are reported the first time for the genus Polygala.

Materials and Methods

M.p.: uncorrected; UV: Perkin-Elmer Lambda 35 UV/VIS spectrometer; NMR: Bruker AV 400 with TMS as internal standard, DMSO-d ₆ or CDCl₃ as solvent, ¹H-NMR at 400 MHz, ¹³C-NMR at 100 MHz, 2D data were obtained using the standard XWIN-NMR 3.1 software package (Bruker); IR: KBr discs; MS: Micromass ZabSpec high-resolution mass spectrometer.

The roots of Polygala caudata were collected in September 2002, from Guizhou Province of China, and authenticated by Dr. Bao-Lin Guo, Peking Union Medical College, Beijing, China. A voucher specimen (No. D 4415) was deposited in the Herbarium of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

Dried and cut roots (27 kg) were refluxed with 95 % and 50 % EtOH, each twice for 2 h. The combined extracts were concentrated under vacuum to leave a residue, which was suspended in H₂O (10 L) and extracted with petroleum (2 L × 5), EtOAc (2 L × 5) and n-BuOH (2 L × 5), respectively. The EtOAc extract (200 g) was chromatographed on silica gel (100 - 200 mesh, 1.5 kg), eluted successively with CHCl₃ (12 L), EtOAc (12 L), acetone (9 L) and MeOH (9 L).

Compound 7 (13 g), yellow needle crystals, was obtained from the CHCl₃ solution (58.5 g) while it was concentrated. The CHCl₃ mother liquor was chromatographed on silica gel (200 - 300 mesh, 1.5 kg), eluting with petroleum/acetone (4 : 1, gradient), whereby 26 fractions (each of 1500 mL) were collected. Fractions 6 - 10 and 12 - 15 were respectively purified by Sephadex LH-20 CC eluted with CHCl₃/MeOH (1 : 1) to yield compound 5 (80 mg) and compound 2 (200 mg). Fractions 16 - 18 were chromatographed on polyamide (100 - 200 mesh, 35 g) eluted with MeOH/H₂O (4 : 1, gradient) in 70 mL fractions. Sub-fractions 5 - 7 were subjected to Sephadex LH-20 CC eluted with CHCl₃/MeOH (1 : 1), and compound 1 (35 mg) was obtained.

The EtOAc residue (90 g) was chromatographed on polyamide (40 - 60 mesh, 400 g) eluting with MeOH/H₂O (4 : 1, gradient). Fractions of 800 mL each were collected to give 50 fractions. Fractions 12 - 22 and 24 - 28 were combined to afford fractions A (18.5 g) and B (5.0 g), respectively, after TLC examination. Fraction A was separated into 34 fractions (600 mL) on a silica gel (200 - 300 mesh, 600 g) column eluted with CHCl₃/MeOH (9 : 1, gradient), compound 4 (1.6 g) was obtained from fraction 12, fractions 3 - 8 of A were re-chromatographed on a silica gel (200 - 300 mesh, 100 g) column with petroleum/acetone (4 : 1) as eluent in 100 mL fractions, compounds 3 (40 mg) and 6 (120 mg) were obtained from sub-fractions 7 - 8 and 11 - 13, respectively. Fraction B was purified by silica gel (200 - 300 mesh, 200 g) CC eluted with petroleum/acetone (4 : 1), fractions of 200 mL were collected, and sub-fractions 4 - 5 afforded compound 8 (100 mg). Each compound was purified by Sephadex LH-20 CC with CHCl₃/MeOH (1 : 1) as eluent.

2-Hydroxy-1,6,7-trimethoxyxanthone (1): white needles, m. p. 246 - 248 °C; UV (MeOH): λ = 244, 322, 370 nm; IR (KBr): ν = 1650, 1620, 1510, 1475, 1425, 1275, 1190, 1130, 1055, 1030 cm^-1; ¹H-NMR (400 MHz, CDCl₃): δ = 7.65 (1H, s, H-8), 7.36 (1H, d, J = 9.2 Hz, H-3), 7.20 (1H, d, J = 9.2 Hz, H-4), 6.86 (1H, s, H-5), 6.01 (1H, s, OH-2), 4.06 (3H, s, OMe-1), 4.01 (3H, s, OMe-6), 3.99 (3H, s, OMe-7); ¹³C-NMR: see Table [3]; EI-MS: m/z (%) = 302 (M⁺, 63), 284 (100); HR-EI-MS: m/z = 302.0787 [M]⁺ (calcd. for C₁₆H₁₄O₆ : 302.0790).

1,4-Dimethoxy-2,3-methylenedioxyxanthone (2): white needles, m. p. 203 - 204 °C; UV (MeOH): λ = 219, 247, 307, 350 nm; IR (KBr): ν = 2935, 1655, 1610, 1485, 1455, 1425, 1325, 1265, 1235, 1060 cm^-1; ¹H-NMR (400 MHz, CDCl₃): δ = 8.28 (1H, dd, J = 8, 1.6 Hz, H-8), 7.66 (1H, ddd, J = 7.6, 7.8, 1.6 Hz, H-6), 7.49 (1H, d, J = 8 Hz, H-5), 7.35 (1H, dd, J = 7.6, 7.6 Hz, H-7), 6.09 (2H, s, OCH₂O), 4.09 (3H, s, OMe-4), 4.08 (3H, s, OMe-1); ¹³C-NMR: see Table [3]. EI-MS: m/z (%) = 300 (M⁺, 100), 285 (14), 272 (48), 257 (43), 239 (29); HR-EI-MS: m/z = 300.0656 [M]⁺ (calcd. for C₁₆H₁₂O₆ : 300.0634).

7-Hydroxy-1,2-dimethoxyxanthone (3): yellow needles, m. p. 231 - 232 °C; UV (MeOH): λ = 242, 262, 320, 385 nm; IR (KBr): ν = 3340, 2940, 1645, 1615, 1575, 1475, 1280, 1225, 1055 cm^-1; ¹H-NMR (400 MHz, DMSO-d ₆): δ = 9.83 (1H, s, OH-7), 7.54 (1H, d, J = 9.2 Hz, H-3), 7.38 (1H, d, J = 9.2 Hz, H-5), 7.36 (1H, d, J = 3.2 Hz, H-8), 7.30 (1H, d, J = 9.2 Hz, H-4), 7.19 (1H, dd, J = 9.2, 3.2 Hz, H-6), 3.80 (3H, s, OMe-2), 3.35 (3H, s, OMe-1); ¹³C-NMR: see Table [3]; EIMS: m/z (%) = 272 (M ⁺, 76), 257 (100), 243 (38), 229 (75); HR-EI-MS: m/z = 272.0688 [M]⁺ (calcd. for C₁₅H₁₂O₅ : 272.0685).

Five known compounds, 2,7-dihydroxy-1-methoxyxanthone (4) [9], 1-methoxy-2,3-methylenedioxyxanthone (5) [10], 7-hydroxy-1-methoxyxanthone (6) [1], euxanthone (1,7-dihydroxyxanthone) (7) [11] and gentitein (1,3,7-trihydroxyxanthone) (8) [4] were identified by comparison of their m. p.s and spectra data (UV, IR, NMR and mass) with those in the literature.

The bioactivity screenings were carried out in vitro. The luminol chemiluminescence method was employed for antioxidation activity assays [12], [13]. The scavenging activity (%) was measured as the scavenging effect on H₂O₂ and the reactive oxygen free radicals produced by macrophage respiratory burst. Ascorbic acid was used as a reference compound. In the vasodilatation assay, the tension relaxation value of the thoracic aorta rings was recorded after the sample was added to the thoracic aorta ring of Wistar rats contracted by KCl, and the dilatation effect (%) was evaluated [14]. Verapamil, a vasodilatation drug, was used as a positive control.

All the procedures involved in animal studies were performed according to the Guide for Care and Use of Laboratory Animals of the National Institutes of Health [15].

Acknowledgements

The authors are grateful to Prof. Chao-Yi Deng, Institute of Forest Science, Xingyi, Guizhou Province, China, for the collection of plant material. They are also indebted to Professor Guang-Hua Du, Institute of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing, China, for the assistance in bioactivity testing.

References

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Dr. Shi-Lin Chen

Department of Applied Biology & Chemical Technology

The Hong Kong Polytechnic University

Hung Hom, Kowloon

Hong Kong

Fax: +852-2364-9932

Email: bcslchen@inet.polyu.edu.hk

References

• 1 Li W K, Chan C L, Leung H W, Yeung H W, Xiao P G. Xanthones from Polygala caudata .  Phytochemistry. 1999;  51 953-8
  CrossrefPubMedSearch in Google Scholar
• 2 Pan M D, Mao Q. Isolation and identification of wubangziside A and B from Polygala caudata Reld et Wils.  Acta Pharm Sin. 1984;  19 899-903
  PubMedSearch in Google Scholar
• 3 Pan M D, Mao Q. Isolation and identification of wubangziside C from Polygala caudata Reld et Wils.  Acta Pharm Sin. 1985;  20 662-5
  PubMedSearch in Google Scholar
• 4 Li W K, Chan C L, Leung H W, Yeung H W, Xiao P G. Xanthones and flavonoids of Polygala caudata .  Pharm Pharmacol Commun. 1998;  4 415-7
  PubMedSearch in Google Scholar
• 5 Mao S L, Liao S X, Wu J H, ling N, Chen H, Liang H Q. et al . Studies on chemical constituents of Polygala arillata Buch-Ham.  Acta Pharm Sin. 1997;  32 360-2
  PubMedSearch in Google Scholar
• 6 Mak N K, Li W K, Zhang M, Wong R NS, Tai L S, Yung K KL, Leung H W. Effects of euxanthone on neuronal differentiation.  Life Sci. 2000;  66 347-354
  PubMedSearch in Google Scholar
• 7 Harborne J B. Phytochemical Methods. Chapman & Hall London; 1973
  Search in Google Scholar
• 8 Frahm A W, Hambloch H. Computer supported structure elucidation of polymethoxy- and polyacetoxyxanthones.  Org Magn Reson. 1982;  19 43-8
  CrossrefPubMedSearch in Google Scholar
• 9 Yang X D, Xu L Z, Yang S L. Xanthones from the stems of Securidaca inappendiculata .  Phytochemistry. 2001;  58 1245-9
  CrossrefPubMedSearch in Google Scholar
• 10 Ghosal S, Basumatari P C, Banerjee S. Extractives of Polygala. Part 8. 1,2,3-Trioxygenated glucosyloxyxanthones from Polygala triphylla .  Phytochemistry. 1981;  20 489-92
  CrossrefPubMedSearch in Google Scholar
• 11 Fujita T, Liu D Y, Ueda S, Takeda Y. Xanthones from Polygala tenuifolia .  Phytochemistry. 1992;  31 3997-4000
  CrossrefPubMedSearch in Google Scholar
• 12 Hu T X, Chen J W, Xu J Y, Lu J Y, Yang Q Y. Effects of polysaccharide-peptide of Coriolus and polysaccharide of Ganoderma on scavenging active oxygen species.  Acta Bioch Biophys Sin. 1992;  24 465-470
  PubMedSearch in Google Scholar
• 13 Feng R T, He W, Ochi H. Experimental studies on antioxidation of extracts from several plants used as both medicines and foods in vitro .  J Chin Med Mat. 2000;  23 690-3
  PubMedSearch in Google Scholar
• 14 Cheng Y W, Kang J J. Mechanism of vasorelaxation of thoracic aorta caused by xanthone.  Eur J Pharmacol. 1997;  336 23-8
  CrossrefPubMedSearch in Google Scholar
• 15 Ministry of Health P. R. China. Guide for the Care and Use of Laboratory Animals 2001
  Search in Google Scholar

Dr. Shi-Lin Chen

Department of Applied Biology & Chemical Technology

The Hong Kong Polytechnic University

Hung Hom, Kowloon

Hong Kong

Fax: +852-2364-9932

Email: bcslchen@inet.polyu.edu.hk